Project Summary Cell survival in the presence of fluctuating environmental signals is critically dependent on rapid changes in gene expression. Chromatin-modifying enzymes are key regulators of genome reprogramming during stress, and aberrant regulation or mutation of these enzymes results in disrupted gene expression programs and inappropriate responses to cellular stress. Such consequences contribute to pathological processes including oncogenesis and aging of human cells. Despite critical roles for chromatin modifiers in these pathways, there are still substantial gaps in our knowledge regarding novel sites of histone modification and their effects on genome regulation, particularly in the presence of diverse stresses encountered in the environment. Our preliminary work has uncovered that the Saccharomyces cerevisiae protein Set4, a potential ortholog of the human protein MLL5, is important for cell survival in oxidative stress and that it is an active histone methyltransferase. The central hypothesis of our work is that Set4 is a stress- regulated methyltransferase that activates a defined gene expression program in response to stress through its lysine methylation activity. Three specific aims are proposed. In Aim I, we will define the substrate specificity of Set4 both in vitro and in cells using biochemical and mass spectrometry based approaches. We will test whether Set4 primarily targets histones, or if it also has additional, non-histone methyl-lysine substrates that contribute to the oxidative stress response. In Aim II, molecular and genetic analysis will be used to determine pathways that regulate Set4 itself in response to oxidative, and other, stresses. Mechanisms by which Set4 controls gene expression will be elucidated through RNA-sequencing analysis and chIP- sequencing of Set4 and its cognate methyl mark in a series of mutants under stress. Aim III will test the hypothesis that the PHD finger of Set4 is required for Set4-depdent stress responses by promoting its localization to chromatin. Biochemical and proteomic assays will determine the histone or non-histone binding partner of the PHD finger of Set4, and targeted molecular and genomic experiments will assess the role of the PHD finger in the localization and activity of Set4 at chromatin. These research aims will provide substantial insight in to the function of a novel epigenetic modifier that we expect to be applicable to its potential human ortholog MLL5, which has been implicated in stem cell maintenance, tumorigenesis and neurodevelopmental disorders. Furthermore, this work will uncover new links between environmental stress and chromatin-based regulation of gene expression, which will be critical to our understanding of how misregulation of the genome by aberrant stress signaling contributes to human disease.